Empirical validation of viral quasispecies assembly algorithms: state-of-the-art and challenges

Abstract

Next generation sequencing (NGS) is superseding Sanger technology for analysing intra-host viral populations, in terms of genome length and resolution. We introduce two new empirical validation data sets and test the available viral population assembly software. Two intra-host viral population ‘quasispecies’ samples (type-1 human immunodeficiency and hepatitis C virus) were Sanger-sequenced, and plasmid clone mixtures at controlled proportions were shotgun-sequenced using Roche's 454 sequencing platform. The performance of different assemblers was compared in terms of phylogenetic clustering and recombination with the Sanger clones. Phylogenetic clustering showed that all assemblers captured a proportion of the most divergent lineages, but none were able to provide a high precision/recall tradeoff. Estimated variant frequencies mildly correlated with the original. Given the limitations of currently available algorithms identified by our empirical validation, the development and exploitation of additional data sets is needed, in order to establish an efficient framework for viral population reconstruction using NGS.

CoVaMa: Co-Variation Mapper for disequilibrium analysis of mutant loci in viral populations using next-generation sequence data

“…However, this imposes certain assumptions about the expected and observed frequencies of distant mutations and the rates of recombination between them (either due sequencing artifacts or the viral replication itself). Indirect approaches also include ‘quasi-species reassembly’ algorithms that aim to reconstruct the multiple variant viral genomes within a population, of which there are multiple strategies (reviewed in [8, 9]).…”

Invertebrate RNA virus diversity from a taxonomic point of view

“…Most important among these is the scarcity or complete lack of biologically relevant data, including the host range, pathological impact on hosts(s) and virion characteristics, for the viral sequences produced using highthroughput methods. Also, in silico assembly of relatively short NGS reads from viral populations can be hindered by the quasispecies structure of RNA virus populations (Prosperi et al, 2013), and could impact on the accuracy of the assembled virus sequences. The ICTV has defined standards for virus annotation (Brister et al, 2010), and for establishing new virus species and higher taxonomic groups (King et al, 2012), in an effort to resolve challenges related to the use of NGS.…”

Rapid identification of pathogens using molecular techniques

“…1 Significant advances in sequencing technology, with costeffective whole-genome sequencing (WGS), [2][3][4][5][6] have allowed us to enhance our understanding of subspecies-level or strain-level differences in bacterial and viral genomes, and have revealed previously unknown differences between clones or quasi-species of microbial pathogens. 1 Significant advances in sequencing technology, with costeffective whole-genome sequencing (WGS), [2][3][4][5][6] have allowed us to enhance our understanding of subspecies-level or strain-level differences in bacterial and viral genomes, and have revealed previously unknown differences between clones or quasi-species of microbial pathogens.…”